PROJECT SUMMARY- PROJECT 3 Myalgic encephalomyelitis, or chronic fatigue syndrome (ME/CFS), is a prevalent and severe disease with symptoms including problems with concentration, memory and sleep, together with musculoskeletal pain. There is no established cause for ME/CFS; neither vaccines nor effective treatment options exist. Therefore, there is an urgent need to advance our understanding of the basic science of this disease as an integral step towards an ultimate cure. Despite our lack of understanding of ME/CFS, substantial evidence implicates immune dysregulation either as an underlying cause or major consequence of the disease. Nevertheless, the identity of specific dysregulated leukocytes (white blood cells, WBC) that are most implicated in ME/CFS is unclear. Project 3, part of our ME/CFS Collaborative Researcher Center application, proposes to elucidate immune dysregulation in ME/CFS by comprehensively investigating gene regulation defects in patients across all types of leukocytes. In Aim I, we propose to use single-cell RNA sequencing (scRNAseq) to interrogate the transcriptomes of leukocytes from peripheral blood collected from a cohort of ME/CFS patients and controls. This novel approach offers multiple advantages over all previous gene regulatory studies of the disease. First, scRNAseq is inherently unbiased with respect to the identity of the specific cell-types examined. Second, scRNAseq generates data with remarkable specificity and statistical power, allowing us, for example, to define the activation state of individual immune cells. Third, our scRNAseq approach will generate simultaneous measurements across the spectrum of cell types in blood. Thus, we will be able to discover coordinate changes in gene regulation between different cell types. In Aim II, we will extend our use of scRNAseq to compare leukocyte profiles from patients and controls post-exercise. This Aim is motivated by the need to better understand aberrant gene regulation in specific leukocytes resulting from exercise in ME/CFS patients, as post-exertional malaise is a defining symptom of the disease. Finally, in Aim III, we will examine possible roles for microRNAs found in extracellular vesicles (ECV-miRNAs) in ME/CFS. Extracellular vesicles are a newly discovered class of particles secreted by donor cells, which deliver cargoes, including miRNAs, to recipient cells, thereby altering the state of the recipient cell. Specifically, we will test the hypothesis that ECV- miRNAs are altered in ME/CFS, examining samples obtained pre- and post- exercise challenge, and examine whether such alterations result in dysregulation of patient immune cells. Thus, the unifying theme of this study is to provide definitive data on gene regulatory changes in leukocytes from ME/CFS patients, and investigate whether ECV-miRNAs contribute to such changes. Because the patient and control cohorts examined are common across this U54 application, we will generate gene regulatory data from leukocytes before and after exercise (Project 3), together with neurological (Project 1) and physiological/molecular data (Project 2). Our comprehensive data will be a key resource not only for ME/CFS Centers, but for all researchers in the field.